Our laboratory focuses on elucidating the coupling of the forces, structure, and dynamics of biologically important macromolecules. The next challenge in structural biology is to understand the physics of interactions between molecules in aqueous solution. The ability to take advantage of the increasing number of available protein and nucleic acid structures will depend critically on establishing the link between structure and interaction energies. A fundamental and quantitative knowledge of intermolecular forces is necessary for understanding the strength and specificity of interactions among biologically important macromolecules that control cellular function and for rationally designing agents that can effectively compete with those interactions associated with disease. Our past results have shown that experimentally measured forces are very different from those predicted by currently accepted theories. We have interpreted the observed forces as indicating the dominating contribution from water structuring energetics. We continue to measure directly forces between biological macromolecules in macroscopic condensed arrays using osmotic stress and x-ray scattering. To investigate directly the role of water in the interaction of individual molecules we also measure and correlate changes in binding energies and hydration accompanying specific recognition reactions of biologically important macromolecules in solution, particularly of sequence specific DNA-protein complexes.[unreadable] The ability to measure forces between biopolymers in macroscopic condensed arrays directly has greatly changed our understanding of how molecules interact at close distances, the last 1-1.5 nm separation. The universality of the force characteristics observed for a diverse variety of macromolecules, including DNA, proteins, lipid bilayers, and carbohydrates, and for the interaction of small solutes with macromolecules has led us to conclude that the energy associated with changes in hydration structure between surfaces at close spacing dominates intermolecular forces. The compaction of DNA in the cell is mediated by highly positively charged proteins such as histones and protamines. DNA packaging by synthetic polycations is central for delivery systems used in gene therapy. Our previous measurements have suggested that the attractive force between DNA helices mediated by highly charged cations is likely due to water structuring rather than electrostatics. DNA helices assembled by these multivalent ions are separated by some 7-10 Angstroms of water depending on the condensing cation, indicating there are both attractive and repulsive forces. In order to more definitively connect attraction and hydration forces, we have combined the osmotic stress x-ray measurements that probe repulsive forces with single molecule, magnetic tweezers experiments designed to probe attractive forces between DNA helices. We have focused on biogenic oligo- and polyamines and trivalent cobalt hexammine. The hydration force formalism makes two very specific predictions that are confirmed by our experiments. First, the repulsive force amplitude that opposes assembly should be the same for a set homologous cations. This force amplitude should be simply connected to the hydration of groups on the DNA surface. We find indeed that the limiting repulsive force is the same for spermidine, spermine, an alkyl hexamine, and even for the alkyl diamine putrescine that does not cause spontaneous DNA assembly. The repulsive force amplitude is quite different for cobalt hexammine that structures water differently. The second prediction is that the ratio of the attractive and repulsive free energies at the equilibrium spacing between helices should be slightly larger than two due to the predicted factor of two difference in exponential decay lengths between attractive and repulsive hydration forces. We calculate the repulsive free energy at the equilibrium spacing from the osmotic stress measurements that give the forces necesary to push helices closer than the equilibrium separation. The magnetic tweezers pulling force necessary to just prevent collapse of a single DNA molecule to the equilibrium spacing gives the net attractive force. Combining this with the osmotic stress measurement of the repulsive free energy, we can calculate the attractive free energy component itself. For all four condensing ions examined the ratio of attractive and repulsive free energies is 2.1, confirming the prediction based on solely hydration forces. [unreadable] Our ultimate goal is to apply the lessons from direct force measurements to the recognition reactions that control cellular processes. We therefore focus on the role of water in specific binding, particularly of sequence specific DNA binding proteins. We measure differences in water sequestered by complexes of sequence specific DNA binding proteins with varied DNA sequences, with particular emphasis on correlating binding energy and water incorporated and on determining the energy necessary to remove hydrating water from complexes. We determine differences in sequestered water among complexes through the effect of changing water activity or, equivalently, osmotic pressure on binding constants and dissociation rates. In order to validate the osmotic stress approach we have now measured the difference in sequestered water between specific and nonspecific complexes of the type II restriction endonuclease, BamHI. X-ray crystal structures for both the BamHI specific and noncognate complexes are known. In contrast to the close interaction of protein and DNA in the specific sequence complex, the nonspecific complex structure shows a gap between the BamHI and DNA major groove surfaces that is large enough to accommodate about 150 waters. Using the osmotic stress technique, we measured the dependence of specific-nonspecific BamHI-DNA binding competition on osmotic pressure. We find that the nonspecific complex sterically sequesters 120 ? 144 more waters than the specific complex in good agreement with the structural data. We also explored the limits of the osmotic stress approach applied to reactions that result in large changes in exposed surface area. [unreadable] The relationship between binding free energy and sequestered water for the binding of bacteriophage lambda Cro repressor protein to a set of DNA operator sequences has now been established. The binding energy of Cro repressor gradually decreases as the optimal DNA sequence is changed as is typical for many DNA-protein complexes. The set of operator sequences examined span a range of 4 Kcal/mole in Cro repressor binding energy. Remarkably, there is a linear relationship between the number of water molecules sequestered and binding free energy. Each extra water molecule incorporated by a noncognate Cro-DNA complex is accompanied by a binding energy decrease of about 0.15 Kcal/mole. Waters and binding energies are directly linked. Previous measurement from another lab showed that heat capacity changes linked to complex formation also vary linearly with binding free energy. Heat capacity changes are thought due to changes in hydration. Combining the data sets suggests that the release of one water molecule contributes 8 cal/mole oK to the heat capacity. This is close to the heat capacity difference between ice and liquid water further suggesting that the incorporated waters are integral to the complex structure. Corresponding changes in enthalpy and entropy for Cro-DNA complexes, however, are very different from the ice-liquid water transition, complicating the analysis. A linear variation would also occur if binding to noncognate sequences is characterized by an equilibrium between discrete structures, e.g., specific and nonspecific binding modes.